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Phrynops Hilarii

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Phrynops Hilarii Vol XXXI (1): 15-22; July 2020 Journal of the Argentine Society of Genetics ANALYSIS OF GENOTOXICITY IN ERITHROCYTES OF TURTLES (Phrynops hilarii) FROM ANTHROPIZED AND NATURAL SITES OF ENTRE RÍOS, ARGENTINA ANÁLISIS DE GENOTOXICIDAD EN ERITROCITOS DE TORTUGAS (Phrynops hilarii) DE SITIOS ANTROPIZADOS Y NATURALES DE ENTRE RÍOS, ARGENTINA Castaño G. V. 1, Cabagna Zenklusen M. 2, Prieto Y. 1, Manzano A. S. 1 ABSTRACT 1 Centro de Investigaciones The micronucleus test (MN) is a biomarker of non-destructive genotoxicity that allows Científicas y Transferencia de Tecnología a la Producción chromosomal damage and other nuclear alterations (NA) to be detected. Phrynops hilarii (CICYTTP-CONICET- UADER), Materi is a freshwater chelonium that inhabits regions of central-northern Argentina. The main y España, Diamante, Entre Ríos, objective was to determine the presence of MN and other NA in erythrocytes of natural Argentina. populations of P. hilarii comparing their frequencies between three sites, two anthropized and one of control (cities of Diamante and Paraná) of Entre Ríos, Argentina, during the 2 Universidad Nacional del Litoral, Facultad de Bioquímica y Ciencias period 2015-2016. Eighteen individuals (six per sampling site) were evaluated at the sites: 1- Biológicas, Paraje el Pozo s/n, Santa PD: Pre-Delta National Park (control), 2- AG: Salto Ander Egg (agroecosystem) and 3- SU: Fe, Argentina. Caleta Club Náutico (urban system). Blood was obtained from the femoral vein. The samples were stained with the May Grünwald-Giemsa method and observed under a microscope with Corresponding author: an immersion objective. Micronucleus (MNF) and nuclear alterations (NAF) frequencies Adriana Silvina Manzano were determined every 1000 erythrocytes observed. A significant difference (p<0.05) was [email protected] found between the PD site and the other sites (AG and SU), both for MNF (p=0.0021) and for ORCID 0000-0002-6862-857X NAF (p=0.0011). The highest frequency values ​​corresponded to the AG site (MNF: 3.33 ± 0.62; NAF: 4.67 ± 0.56). Finally, biomonitoring with P. hilarii was useful, so it could be considered as a bioindicator species to assess the quality of Argentina’s environments. Genotoxicity biomarkers, micronucleus test, nuclear alterations Cite this article as: Key words: Castaño G. V., Cabagna Zenklusen M., Prieto Y., Manzano A. S. 2020. ANALYSIS OF GENOTOXICITY IN RESUMEN ERITHROCYTES OF TURTLES (Phrynops hilarii) FROM ANTHROPIZED AND El test de micronúcleos (MN) es un biomarcador de genotoxicidad no destructivo que permite NATURAL SITES OF ENTRE RÍOS, detectar daño cromosómico y otras alteraciones nucleares (AN). Phrynops hilarii es un quelonio ARGENTINA. BAG. Journal of Basic de agua dulce que habita regiones del centro-norte de Argentina. El objetivo principal fue and Applied Genetics XXXI (1): 15-22. determinar la presencia de MN y otras AN en eritrocitos de poblaciones naturales de P. hilarii comparando sus frecuencias entre tres sitios, dos antropizados y uno de control (ciudades de Diamante y Paraná) de Entre Ríos, Argentina, durante el periodo 2015-2016. Dieciocho Received: 10/22/2019 individuos (seis por sitio de muestreo) fueron evaluados en los sitios: 1- PD: Parque Nacional Revised version received: 03/03/2020 Pre-Delta (control), 2- AG: Salto Ander Egg (agroecosistema) y 3- SU: Caleta Club Náutico Accepted: 03/15/2020 (sistema urbano). Se extrajo sangre de la vena femoral. Las muestras se tiñeron con el método General Editor: Elsa Camadro May Grünwald-Giemsa y se observaron bajo un microscopio con el objetivo de inmersión. Las frecuencias de micronúcleos (FMN) y alteraciones nucleares (FAN) se determinaron cada DOI: 10.35407/bag.2020.31.01.02 1000 eritrocitos observados. Se encontró diferencia significativa (p<0,05) entre el sitio PD y ISSN online version: 1852-6233 los otros sitios (AG y SU), tanto para FMN (p=0,0021) como para FAN (p=0,0011). Los valores de las frecuencias más altos correspondieron al sitio AG (FMN: 3,33±0,62; FAN: 4,67±0,56). Finalmente, el biomonitoreo con P. hilarii fue útil, por lo que podría considerarse como especie bioindicadora para evaluar la calidad de los ambientes de Argentina. Palabras clave: biomarcadores de genotoxicidad, prueba de micronúcleos, alteraciones nucleares Available online at www.sag.org.ar/jbag ARTICLE 2 - RESEARCH 15 Genotoxicidad en Eritrocitos de Tortugas Hernández Guzmán et al., 2015). Se consideran como INTRODUCCIÓN AN las siguientes características descriptas por Carrasco El test de micronúcleos (MN) es un biomarcador de et al. (1990): 1- núcleo escotado (KN): invaginación genotoxicidad no destructivo que puede aplicarse en relativamente pequeña en la membrana nuclear y distintos tipos de células (epiteliales, sanguíneas, cromatina no condensada; 2- núcleo mellado (EN): sexuales, entre otras) y organismos (animales, vegetales presencia de una muesca en el núcleo; 3- lóbulo o brote y humanos), para detectar daño cromosómico (Pastor nuclear (LN): morfología semejante a un MN conectado Benito, 2002; Poletta, 2011; Lajmanovich et al., 2012; o unido al núcleo; 4- célula binucleada (BN): célula Caraffa et al., 2013). Este test, es un método sensible y con presencia de dos núcleos de tamaños semejantes o rápido que puede realizarse tanto en ensayos de laboratorio similares. El mecanismo por el cual se producen las AN como a campo, sin necesidad de sacrificar al individuo en no es conocido con claridad. López González et al. (2017) evaluación para la obtención de la muestra (Lajmanovich mencionaron que cuando la célula detecta una región et al., 2012; Zapata Restrepo et al., 2017). Esto es de gran de ácido desoxirribonucleico (ADN) afectada, se inicia importancia, ya que puede emplearse en sucesivos un proceso de reparación y eliminación de la cromatina. muestreos y en especies con escaso número de individuos La región alterada se mueve a la periferia del núcleo y o en peligro de extinción (Lajmanovich et al., 2012). se elimina por exocitosis. Antes de que el proceso de Los MN son masas de cromatina con forma de pequeños reparación y eliminación de la cromatina culmine, la núcleos cercanos al núcleo principal en las células de la membrana nuclear presenta algunas imperfecciones, interfase, pudiendo originarse de manera espontánea ocasionando AN. o como respuesta a la acción de ciertos agentes (Pastor Los agentes genotóxicos son sustancias que pueden Benito, 2002) como pueden ser los hidrocarburos actuar de forma directa o indirecta sobre el material (Barsiené y Andreikénaité, 2007), metales (Paolín et al., genético (ADN) y provocar efectos a concentraciones 2010), plaguicidas (Pastor Benito, 2002; Lajmanovich subletales (Gutiérrez, 2013). Estos agentes pueden ser et al., 2014), fármacos (Arranz Gutiérrez, 2016), entre liberados al ambiente por distintas actividades humanas, otros. Cuando esto ocurre, el material genético queda ya sean productivas como la minería, la agricultura y excluido de los núcleos de las nuevas células durante la distintos procesos industriales o por actividades urbanas anafase mitótica, dando lugar a formaciones redondas como el vertido de efluentes cloacales, emisiones en el citoplasma de la célula hija. El material genético vehiculares, aguas residuales hospitalarias y de desprendido puede tener dos orígenes: 1- derivar de estaciones de combustible, entre otras (Paz et al., 2008; cromosomas enteros (efecto aneugénico), donde el Zuluaga Quintero et al., 2009; Gutiérrez, 2013; González daño genotóxico ha afectado a proteínas del cinetocoro, Torres et al., 2015). Para evaluar el riesgo potencial de al centrómero o al huso mitótico, generando un retraso poblaciones naturales expuestas a agentes genotóxicos, mitótico y un desequilibrio en la distribución de los son utilizadas especies indicadoras o centinelas y cromosomas, 2- pueden formarse de fragmentos distintos biomarcadores, ya que suministran señales de cromosómicos acéntricos que han sido excluidos de alarma temprana ante la presencia a tóxicos (Cabagna los núcleos de las nuevas células (efecto clastogénico) Zenklusen, 2012; Caraffaet al., 2013). durante la anafase mitótica (Cabagna Zenklusen, 2012). Los reptiles son organismos muy sensibles a cambios Numerosos trabajos en fauna han aplicado el test de en las condiciones del ambiente, por lo que en muchos MN en eritrocitos, para evaluar a través de su frecuencia casos fueron utilizados como indicadores o centinelas de (FMN) los efectos provocados por distintos agentes en contaminación, debido a que presentan características el material genético y en la división celular (Pollo et como: poblaciones persistentes en diferentes hábitats al., 2012; Caraffa et al., 2013; Latorre et al., 2015). Para (acuáticos y terrestres), longevidad, posición alta en la determinar el potencial de una especie como organismo cadena alimentaria, alta sensibilidad a contaminantes, bioindicador o centinela se utiliza como valor referencia amplia distribución geográfica y fidelidad al sitio la frecuencia basal de micronúcleos (FBMN). Este (Gardner y Oberdorster, 2005; Poletta et al., 2008; valor refiere al número de MN provocados de manera Poletta et al., 2013). Dentro de estos, las tortugas han espontánea como resultado de procesos normales de sido propuestas como organismos bioindicadores y/o replicación y/o división celular (Cabagna Zenklusen, centinelas para evaluar su exposición a sustancias 2012; Latorre et al., 2015). tóxicas (Andreani et al., 2007; De Solla et al, 2007; De Otras alteraciones nucleares (AN) pueden analizarse Solla et al., 2008; Latorre
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